Targeting of herpes simplex virus to specific receptors

ABSTRACT

The invention relates to engineered Herpes simplex virus (HSV) particles that are targeted to one or more specific binding pair members, such as receptors. Also, recombinant vectors for producing such HSV particles are provided. By reducing the affinity of HSV for its natural receptor(s) and increasing the affinity for a selected receptor, the HSV particles of the invention are useful for targeting cells that express the selected receptor, which itself may be a product of genetic engineering. The ability to selectively target cells render the HSV particles. particularly useful in selectively diagnosing, treating, and imaging cells bearing the selected binding pair member, such as a receptor. The invention also provides for polynucleotide-based therapy to cells bearing the selected binding pair member such as a receptor.

GOVERNMENT INTERESTS

The U.S. Government owns rights in the invention pursuant to NationalCancer Institute grant number 1PO1 CA71933.

This application is a U.S. national phase of PCT/US06/33865 filed Aug.30, 2006, which is a continuation in part of U.S. application Ser. No.11/215,636 , filed Aug. 30, 2005, now U.S. Pat. No. 7,550,148, which isa continuation-in-part application of U.S. Ser. No. 10/530,774, filedApr. 7, 2005 with a 371 completion date of Nov. 17, 2005, now U.S. Pat.No. 7,501,126, which is a U.S. national phase of PCT/US03/31598 filedOct. 6, 2013, which claims the priority benefit of U.S. Ser. No.60/416,716, filed Oct. 7, 2002.

BACKGROUND OF THE INVENTION

A steady rate of healthcare advances has led to continuing improvementin the health and quality of life for humans and animals. Nevertheless,a variety of diseases, disorders, and conditions have largely eluded thebest efforts at prevention or treatment. Chief among these maladies isthe loss of cell-cycle control that frequently results in theundesirable cell proliferation characteristic of cancer in its manyforms, such as malignant glioma. Malignant gliomas are devastating braintumors that afflict animals such as humans. The average life span afterdiagnosis is less than one year and few patients have been reported tosurvive five years. Furthermore, none of the conventional anti-cancertherapies has been successful in significantly prolonging the lifespanof patients with this disease. In recent years there have been numerousattempts to use genetically engineered herpes simplex viruses (HSV) asoncolytic agents to treat malignant gliomas. Because wild-type virusesare highly virulent, the viruses used in preclinical evaluations and inphase-1 clinical studies have been thoroughly attenuated. While severaldeletion mutants have been tested, the mutants that reached clinicaltrials lacked the γ₁34.5 gene encoding infected cell protein 34.5(ICP34.5) and optionally, the U_(L)39 gene encoding the large subunit ofribonucleotide reductase.

These attenuated HSV viruses, however, have been imperfectly engineeredas oncolytic agents. One advantage of these mutant viruses is that theyhave a significantly reduced capacity to replicate in normal,non-dividing cells in vivo. Viral ribonucleotide reductase is anessential gene for viral replication in resting cells and, hence, theU_(L)39 mutant virus is dysfunctional in the normal environment of thecentral nervous system (Simard et al. 1995). The major function ofICP34.5 is to preclude the shutoff of protein synthesis caused byactivation of protein kinase R in infected cells. Once activated, thisenzyme phosphorylates the α subunit of translation initiation factor 2(eIF2α), resulting in complete cessation of translation. Mutants lackingthe γ₁34.5 genes are highly attenuated because the lytic life cycle iscompletely blocked in an interferon⁺ cellular background. In contrast,γ₁34.5 mutants are nearly as virulent as wild-type virus in mice lackinginterferon receptor. Although mutants deleted in both γ₁34.5 and U_(L)39are not significantly more attenuated than those lacking the γ₁34.5genes, such mutants do provide added insurance in the form of a reducedrisk of reversion.

A significant disadvantage of these mutant HSV viruses is their poorreplication, even in dividing cells. In experimental animal systems, themutant viruses do not exhibit sustained lytic life cycles, with the lossof a potentially amplified response to a given therapeutic dose of thevirus that would be expected upon re-infection of tumor cells by themultiplied viral progeny. Consequently, maximum killing of tumors cellsrequires high doses of virus. Given the poor growth of these mutant HSVviruses, even in dividing cells, production of virus pools large enoughto yield efficacious inocula of >10⁹ plaque forming units (PFU) hasremained a major obstacle. Moreover, indiscriminate binding of virus tonon-tumor cells further diminishes the effectiveness of HSV virusdosages because mis-targeted viral particles do not contribute to thedesired beneficial therapeutic effect of tumor cell destruction. Oneapproach to overcoming these obstacles is to achieve a more thoroughunderstanding of the HSV lytic life cycle and thereby facilitate thedevelopment of HSV mutants tailored for use as targeted therapeuticagents, such as targeted oncolytic agents.

HSV enters host cells using a two-step mechanism. The first step ofentry is HSV attachment to the cell surface. This step is initiated byglycoproteins B and C (gB and gC), which project from the viralenvelope, attaching to heparan sulfate proteoglycans on host cellsurfaces. The gB and gC domains interacting with heparan sulfate havebeen mapped at the sequence level (Laquerre et al. 1998). Following thisinitial attachment, viral glycoprotein D (gD) interacts with one ofseveral receptors. Of these gD receptors, two are particularly importantfor entry (Spear et al, 2000). One receptor, designated HveA (formerly,HveM), is a member of the family of receptors for tumor necrosisproteins. A second receptor, designated Nectin-1 (HveC), is a member ofthe nectin family of proteins, structurally related to theimmunoglobulin superfamily, which serve as intercellular connectors(Campadelli-Fiume et al. 2000). The second step of HSV entry into a cellis fusion of the viral envelope with the plasma membrane of the cell. Toeffect fusion, gD, when bound to its receptor, recruits glycoproteins B,H and L, which results in fusion of the envelope with the plasmamembrane.

Additional understanding of HSV infection has come from recent studiesthat have lent significance to an old observation that gD interacts withthe cation-independent mannose 6 phosphate receptor, contributing to theaccumulation of HSV in endosomes. Endocytosis of viral particles resultsin particle degradation by lysosomal enzymes, but the cells succumb as aconsequence of the degradation of cellular DNA by lysosomal DNase. HSVgD blocks this apoptotic pathway to cell death through its interactionwith the mannose 6 phosphate receptor. Thus, gD interacts with HveA,nectins, the mannose 6 phosphate receptor, and at least one of thecomplex of viral glycoproteins involved in the fusion of HSV with theplasma membrane.

In an attempt to target HSV-1 infection to specific cells, a recombinantHSV having a chimeric protein comprising gC and erythropoietin (EPO) onits surface was constructed. Although the recombinant virus bound tocells expressing EPO receptor and endocytosis of the virus occurred,successful infection of these EPO-receptor expressing cells did notoccur.

Accordingly, a need continues to exist in the art for viral therapeuticagents exhibiting improved targeting capacities while retainingsufficient capacity to infect to be therapeutically useful. Ideally,suitable viruses would be therapeutic agents, such as oncolytic agents,themselves as well as providing a targeting vehicle or vector for thecontrolled delivery of polynucleotide coding regions useful astherapeutic agents. Another need in the art is for targeted agentsuseful in diagnostic applications as, e.g., imaging agents or targetedvehicles for imaging agents.

SUMMARY

The invention satisfies the aforementioned need in the art by providingviral forms suitable for use as therapeutic and diagnostic agentsthemselves, as well as providing a ready vehicle for the delivery oftherapeutic or diagnostic polynucleotide coding regions to cells. Theseviral forms are modified viruses of the Herpesviridae family of viruses,and are preferably derived from herpes simplex virus type 1 or type 2.The invention provides a method of making virus particles with a novelligand (or binding pair member), and making said particles totallydependent on a receptor of the ligand (or binding pair member) for entryinto targeted cells.

Disclosed herein are methods to modify the surface of, e.g., an HSVvirus particle in a manner that targets the virus to a specific receptorpresent on the surface of a cell of choice, typically a cell in need oftherapy or a cell whose presence provides information of diagnosticvalue. The invention provides viral particles, e.g., HSV particles,having a reduced affinity for their natural cell-surface receptor(s),and methods for producing and using such particles, which minimizes oreliminates the problem of reduced efficiency associated with themis-targeting of therapeutic and diagnostic viruses. Additionally, theinvention provides viral particles, e.g., HSV particles, that exhibitspecific affinity for a cell surface component that is not a naturalviral receptor and that is present solely or predominantly on a giventarget cell, as well as methods for producing and using such viruses.Modified viral particles (e.g., HSV) having increased affinity for acell surface component associated with one or more target cells exhibitimproved targeting capabilities relative to known viral particles. Themodified HSV particles have reduced indiscriminate binding, therebyminimizing sequestration of viral dosages away from the target cells.The invention further provides modified viral particles, such asmodified HSV particles, that have both a reduced affinity for naturalviral receptors and an increased affinity for a cell surface componentassociated with a particular target cell(s), with the modified viralparticle effectively recruiting that cell surface component for use as aviral receptor. Other benefits of the modified viruses are describedherein and will be apparent to those of skill in the art upon review ofthis disclosure.

In one aspect, the invention provides a recombinant herpes simplex virus(HSV) particle having at least one protein on its surface, comprising:(a) an altered gD, wherein the alteration reduces binding of gD to oneor more of its cellular receptors, said alteration comprising (i) aheterologous peptide ligand on the surface of the recombinant HSVparticle forming a fusion protein with the altered gD; and (ii) an aminoacid alteration; wherein said recombinant HSV particle preferentiallybinds to cells expressing a binding partner to said heterologous peptideligand. In some embodiments, these particles preferentially bind totarget cells (cell expressing a binding partner) in whole or part due tothe greater frequency of the binding partner on the surface of the cellrelative to any natural HSV binding proteins on the surface of thatcell. In some embodiments, the recombinant HSV particle furthercomprises an altered viral surface protein, wherein the alterationreduces binding of the viral surface protein to a sulfated proteoglycan.Such recombinant herpes simplex virus (HSV) particles comprise a virussurface protein altered to reduce the wild-type level of binding of thatprotein to a sulfated proteoglycan on the surface of a cell and analtered gD. The altered gD exhibits a reduced binding to one or more ofthe natural cellular receptors for gD; the altered gD is also fused to aheterologous peptide ligand (or binding pair member) having a bindingpartner, e.g., a peptide ligand receptor, found on the surface of acell. Stated in the alternative, this aspect of the invention provides arecombinant herpes simplex virus (HSV) particle having at least oneprotein on its surface, comprising: (a) an altered viral surfaceprotein, wherein the alteration reduces binding of the viral surfaceprotein to a sulfated proteoglycan; and (b) an altered gD, wherein thealteration reduces binding of gD to one or more of its cellularreceptors, the alteration comprising (i) a heterologous peptide ligand(or binding pair member) on the surface of the recombinant HSV particleforming a fusion protein with the altered gD; and (ii) an amino acidalteration; wherein the recombinant HSV particle preferentially binds tocells expressing a binding partner to the heterologous peptide ligand(or binding pair member).

The invention comprehends a recombinant HSV particle wherein the aminoacid alteration is selected from the group consisting of an amino aciddeletion, an amino acid substitution and an amino acid insertion. Apreferred site for the amino acid alteration is amino acid position 34of gD. Exemplary recombinant HSV particles according to the inventioninclude HSV R5141 and HSV R5161, each described below.

Contemplated amino acid alterations include insertions or deletions of1-10 amino acids, such as insertions or deletions of 1-5 amino acids.Exemplary insertions occur immediately upstream (N-terminal) ordownstream (C-terminal) to amino acid position 34 of gD. Exemplarydeletions include amino acid position 34 of gD. For alterationscomprising amino acid substitutions, 1-10 amino acids are substituted,such as substitutions of 1-5 amino acids. Non-contiguous (dispersed) orcontiguous amino acid substitutions are contemplated. In someembodiments, conservative amino acids known in the art are substituted.Exemplary amino acid substitutions include single amino acidsubstitutions for the Serine at position 34 of gD. Preferably,substitutions for Ser34 will be conservative as, for example, an aminoacid substitution of V34S in gD.

The altered gD, moreover, reduces binding of the recombinant HSVparticle to at least one HSV entry mediator (Hve) cell-surface protein,such as an Hve selected from the group consisting of HveA (formerly,HveM) and Nectin-1 (HveC). Further, the recombinant HSV particles of theinvention include particles wherein the altered viral surface protein isselected from the group consisting of gB and gC. In some embodiments,the altered viral surface protein, preferably selected from the group ofgB and gC, forms a fusion protein with a heterologous peptide ligand. Insome embodiments, the binding partner is a cell surface receptor for theheterologous peptide ligand.

The preferential binding of the recombinant HSV particles of theinvention results in a detectable variation in effective binding of theparticle to the cells being compared. By “effective binding” is meanteither sufficiently stable binding to permit detection of binding orbinding sufficient to result in productive infection of the cell. Inpreferred embodiments, the preferential binding is such that therecombinant HSV particles bind only to one of the cell types beingcompared (e.g., cancer cells compared to healthy cells of the sametype). Suitable cells include any hyperproliferative cell type, such asa cancer cell. A cancer cell, in turn, includes a tumor cell, e.g., amalignant gliomal cell.

The invention comprehends recombinant HSV particles wherein theheterologous peptide ligand (or binding pair member) is any ligand (orbinding pair member) for which a cell surface binding partner exists.Preferably, heterologous peptide ligands have specific cell surfacebinding partners, e.g., ligand receptors, that are preferentiallyexhibited on the surface of a target cell. More preferably, the cellsurface binding partner is only exhibited on the surface of a targetcell, when compared to the cells in an organism containing the targetcell. Exemplary heterologous peptide ligands that include cytokines,such as IL13, and fragments, variants and derivatives thereof, providedthat the ligand retains the capacity of binding to a cell-surfacebinding partner. An exemplary binding pair member contemplated assuitable for each aspect of the invention is a single-chain antibody,for which a binding partner would include an antigen thereof, or afragment, derivative or variant thereof that retains the capacity tobind to the single-chain antibody.

Another aspect of the invention is drawn to the recombinant HSV particledescribed above, wherein a polynucleotide encoding the fusion protein isjoined to a heterologous coding region for a leader sequence. In thiscontext, “heterologous” means that the leader sequence is not foundnaturally associated with the upstream or 5′ coding region participatingin the fusion. Exemplary leader sequences include HSV leader sequences,e.g., an HSV gD leader sequence. In a related aspect, the inventionprovides the recombinant HSV particle described above, wherein apolynucleotide encoding the fusion protein is joined to a heterologousexpression control element, such as a heterologous promoter (a promoternot naturally found in association with the polynucleotide coding regionfused upstream or 5′ in the fusion), a heterologous enhancer, orexpression factor binding site known in the art.

Another aspect of the invention provides a pharmaceutical compositioncomprising a recombinant HSV particle described above and apharmaceutically acceptable carrier, diluent, or excipient. Anypharmaceutical carrier, diluent or excipient known in the art iscontemplated. A related aspect of the invention provides a kitcomprising the pharmaceutical composition and a set of instructions foradministering the composition to a subject in need. In each of theseaspects of the invention, i.e., the pharmaceutical compositions and thekits, the heterologous peptide ligands (or binding pair members) andcell-surface binding partners described in the context of describing therecombinant HSV particles are contemplated.

Yet another aspect of the invention provides a method of targeting arecombinant HSV particle to a cell comprising (a) identifying a bindingpair member, such as a ligand for a ligand binding partner, exhibited onthe surface of a target cell; and (b) creating an HSV particle asdescribed herein, wherein the ligand or, more generally, the bindingpair member, binds to the binding partner exhibited on the surface ofthe target cell. In some preferred embodiments of this aspect of theinvention, the altered viral surface protein is selected from the groupconsisting of gB and gC. In some embodiments, the alteration to gDreduces binding of gD to at least one cellular receptor for gD selectedfrom the group consisting of HveA and Nectin-1. In preferredembodiments, the altered gD has a conservative substitution at position34 of gD, such as a V34S substitution. A second fusion protein, joiningthe ligand (or binding pair member) and either of gB or gC, is alsocontemplated in some embodiments. Suitable cells for targeting includeany hyperproliferative cell, such as a cancer cell, including tumorcells (e.g., malignant gliomal cells). Any of the heterologous peptideligands (or binding pair members) and cell-surface binding partnersdescribed above in the context of describing the recombinant HSVparticles is suitable for use in the method.

Another aspect of the invention is drawn to a method of imaging a cellcomprising: (a) contacting the cell with a recombinant HSV particle asdescribed above, the recombinant HSV particle further comprising acoding region for a marker protein; and (b) detecting the presence ofthe marker protein. Any type of cell exhibiting a cell-surface bindingpartner for a ligand (or binding pair member) fusible to HSV gD issuitable for use in this aspect of the invention, such as a cancer cell.Using cancer cells as an example, the method is useful provided that thebinding partner is present at a higher number on the cancer cell ascompared to a non-cancerous cell of the same type. Any known markerprotein is useful in this aspect of the invention, e.g., a markerprotein selected from the group consisting of thymidine kinase, greenfluorescent protein, and luciferase. In preferred embodiments, thealtered gD exhibits an amino acid substitution of V34S. Any of theheterologous peptide ligands (or binding pair members) and cell-surfacebinding partners described above in the context of describing therecombinant HSV particles is suitable for use in the method.

Another aspect of the invention provides a method of treating acell-based disease comprising delivering a therapeutically effectiveamount of a recombinant HSV particle as described herein to a subject inneed. A related aspect is the use of a recombinant HSV particle asdescribed above in the preparation of a medicament for the treatment ofa cell-based disease. A therapeutically effective amount of arecombinant HSV particle is that amount that produces the desiredtherapeutic effect, as would be understood and readily determinable bythose of skill in the art. Any cell-based disease known or reasonablysuspected to be amenable to treatment with a specifically targeted HSVis contemplated, e.g., a cell hyperproliferation disease such as cancer.Any of the heterologous peptide ligands (or binding pair members) andcell-surface binding partners described above in the context ofdescribing the recombinant HSV particles is suitable for use in themethod.

In a related aspect, the invention provides a method of ameliorating asymptom associated with a disease comprising administering atherapeutically effective amount of a recombinant HSV particle describedabove to a subject in need. Another aspect is drawn to the use of arecombinant HSV particle as described above in the preparation of amedicament for ameliorating a symptom associated with a disease in asubject in need. Again, any disease known or reasonably suspected tohave a symptom amenable to application of a specifically targeted HSV iscontemplated, including any disease characterized by hyperproliferativecells, such as cancer. Any of the heterologous peptide ligands (orbinding pair members) and cell-surface binding partners described abovein the context of describing the recombinant HSV particles is suitablefor use in the method.

Another aspect of the invention is directed to a method of delivering atherapeutically useful peptide to a cell comprising: (a) inserting atherapeutically useful polynucleotide, such as an expression controlelement, an rDNA, or a coding region for a therapeutically usefulpeptide, into the DNA of a recombinant HSV particle as described above,thereby producing a recombinant HSV clone; and (b) delivering atherapeutically effective amount of the recombinant HSV clone to thecell. In a related aspect, the invention provides for the use of arecombinant HSV clone comprising a recombinant HSV particle according toclaim 1 in the preparation of a medicament for delivering atherapeutically useful peptide to a cell comprising inserting a codingregion for a therapeutically useful peptide into the DNA of therecombinant HSV particle, thereby producing the recombinant HSV clone.Each of the method and use comprehends delivery of the recombinant HSVclone in vivo, ex vivo, or in vitro. Any of the heterologous peptideligands (or binding pair members) and cell-surface binding partnersdescribed above in the context of describing the recombinant HSVparticles is suitable for use in the method or use.

Another aspect of the invention provides a method of killing a targetcell, comprising contacting the target cell with a recombinant HSVparticle as described above. A related aspect is the use of arecombinant HSV particle as described above in the preparation of amedicament for killing a target cell by contacting the target cell withthe recombinant HSV particle. In preferred embodiments of either themethod or the use, the recombinant HSV particle has an altered gD inwhich the V34S substitution is found. Any of the heterologous peptideligands (or binding pair members) and cell-surface binding partnersdescribed above in the context of describing the recombinant HSVparticles is suitable for use in the method or use.

In each of the above-described aspects of the invention, it is preferredthat gD, or a portion thereof, maintains its membrane fusion properties,but has reduced capacity to bind HveA and/or Nectin-1.

Other features and advantages of the invention will be better understoodby reference to the brief description of the drawing and the descriptionof the illustrative embodiments that follow.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. Schematic representation of the HSV-1 (F) genome and genemanipulations in glycoprotein C (gC) (FIG. 1A), glycoprotein B (gB)(FIG. 1B), and glycoprotein D (gD) (FIG. 1C). Line 1, sequencearrangement of the HSV-1 genome. The rectangular boxes represent theinverted repeat sequences ab and b′a′ flanking the unique long (U_(L))sequence, and inverted repeat c′a′ and ca flanking the unique short(U_(S)) sequence. Line 2, sequence arrangement of domains of theglycoprotein C; the signal peptide (SP) domain and heparan sulfate(HS)-binding domain of gC are highlighted. Line 3, human IL13 withsignal peptide that replaced the N-terminal segment of 148 amino acidsof gC. Line 4, sequence arrangement of the poly-lysine domain of gB.Line 5, schematic representation of a recombinant HSV-1(F) genome, inwhich the N-terminal domain of gC was replaced with IL13 and thepolylysine domain (from codon 68 to codon 77) of gB was deleted. Line 6,sequence arrangement of glycoprotein J (gJ), glycoprotein D (gD), andglycoprotein I (gI) in U_(S). Line 7, replacement of gD with theimmediate early promoter of CMV in order to enable the expression of gI.Line 8, schematic representation of recombinant HSV-1(F) genome, inwhich the N-terminal domain of gC was replaced with IL13, thepoly-lysine domain of gB was deleted, and IL13 was inserted after aminoacid 24 of gD (FIG. 1D). Line 9, a polylinker XhoI-BgIII-EcoRI-KpnI wasinserted after amino acid 24 of gD, with IL13 inserted into the XhoI andKpnI sites of gD.

FIG. 2. Amino acid sequence alignment of IL13-gC, IL13-gD junctionsequence, and the HS binding domain of gB. FIG. 2A. The amino-terminalsequence of IL13-gC chimeric protein (SEQ ID NO:22). The sequencesupstream and downstream of the HS binding site portion are shown. IL13was inserted between the two restriction enzyme sites that areunderlined. FIG. 2B. The domain of the gB open reading frame (i.e., ORF)from which the poly lysine [poly(K)] sequence was deleted (SEQ IDNO:23). The underlined sequences (codons 68-77) were not present in gBamplified from R5107. FIG. 2C. The amino-terminal sequence of IL13-gD(SEQ ID NO:24). The first underlined sequence identifies the gD signalpeptide. IL13 (bracketed by arrows) was inserted between residues 24 and25 (underlined) of gD, between the XhoI and KpnI restriction enzymesites.

FIG. 3. Verification of R5111 viral DNA by PCR. Photographs ofelectrophoretically separated PCR products amplified directly from theplaques picked from Vero (FIG. 3A) and HEp-2 (FIG. 3B) cells. Viral DNAswere extracted as described in Example 1 and subjected to PCR with“IL13” primers from the IL13 ORF and IL 3-gD primers, which bracketedIL13 and the gD ectodomain.

FIG. 4. Photograph of electrophoretically separated proteins fromlysates of cells infected with R5111 reacted with antibody to gC, gD orIL13. HEp-2 cells grown in 25 cm² flasks were exposed to 10 PFU of HSV-1or R5111 per cell. The cells were harvested 24 hours after infection,solubilized, subjected to electrophoresis in 10% denaturingpolyacrylamide gels, electrically transferred onto a nitrocellulosesheet, and exposed to a monoclonal antibody against gD (FIG. 4A), gC(FIG. 4B) or IL13 (FIG. 4C), respectively. The protein bandscorresponding to the gC, IL13-gC fusion protein, gD and the IL13-gDfusion protein are indicated. IL13-gC was the same size as native gC, asexpected.

FIG. 5. HA-tagged IL13Rα2 expression from individual clones of stabletransfectants of the J1.1 cell line. The individual clones wereamplified as described in Example 1. Cells were harvested from 25 cm²flasks, solubilized, and subjected to electrophoresis in 12% denaturingpolyacrylamide gels, electrically transferred onto a nitrocellulosesheet, and exposed to a polyclonal antibody to HA tag.

FIG. 6. Diagram of the pgD-vector.

FIG. 7. Schematic representation of the HSV-1 (F) genome and geneticstructure of R5141 and R5144.

DETAILED DESCRIPTION

The invention provides benefits that will improve the health andwell-being of animals such as man by providing a targeted approach tothe treatment of a variety of conditions and diseases that currentlyimpair health, resulting in significant economic burdens usingconventional treatments. In providing modified viral particles havingcontrollable targeting capacities, the diagnostic and therapeuticbenefit of the viruses themselves can be delivered with greaterprecision to particular cells. Additionally, these viral particles canbe used as targeting vehicles for the delivery of a wide variety oftherapeutic and diagnostic biomolecules, such as polynucleotidesencoding therapeutic or diagnostic peptides.

Beyond the modified viral particles, the invention provides methods formaking such therapeutic and diagnostic agents as well as methods forusing the agents to diagnose or treat a variety of diseases andconditions, such as tumorigenic disease (e.g., gliomas). To facilitatean understanding of the invention and all of its aspects, illustrativeembodiments are described below. The descriptions of these illustrativeembodiments are not meant to limit the invention to the embodimentsdisclosed herein. In light of the description, one of skill in the artwill understand that many changes and modifications can be made to theillustrative embodiments and still remain within the invention. Theillustrative embodiments are disclosed using as an exemplary virus amember of the Herpesviridae family of viruses, herpes simplex virus(HSV).

As noted above, HSV-1 and HSV-2 are members of the family of virusesknown as the Herpesviridae, whose structures are well known in the art.The targeting methods of the invention are applicable to any member ofthe Herpesviridae and are not limited to the exemplary embodimentsdescribed in the examples. Furthermore, a large number of recombinantHSV viruses are known in the art. Such viruses may contain one or moreheterologous genes. Also, such viruses may contain one or more mutatedHSV genes, for example, mutations that render the virusreplication-deficient or affect the virulence of the virus in one ormore cell types.

Examples of recombinant HSV containing a heterologous gene and methodsof making and using such viruses are described in U.S. Pat. No.5,599,691 (incorporated herein by reference in its entirety). Preferredheterologous genes include genes encoding marker proteins. Markerproteins, such as green fluorescent protein, luciferase, andbeta-galactosidase, allow detection of cells expressing the protein. Inother embodiments, the heterologous gene encodes an enzyme thatactivates a prodrug thereby killing adjacent uninfected cells. In yetother embodiments, the heterologous gene encodes a protein that affectsthe immune response, such as interleukin 12 (IL-12). Such proteins thatactivate the immune response against a tumor are particularly useful.

In one aspect, the invention relates to altering the surface of an HSVparticle to target the virus to a specific receptor. By creating afusion protein comprising a portion of gD and a ligand (or binding pairmember), the virus is targeted to a cell having a cell surface receptorthat binds the ligand (or binding pair member). In preferredembodiments, one or more HSV surface proteins, such as gB (SEQ ID NOs:27and 28), gC (SEQ ID NOs:29 and 30), or gD (SEQ ID NOs:25 and 26), arealtered to reduce binding to natural HSV receptors.

“Alterations” of the surface of an HSV particle or HSV surface proteininclude insertions, deletions, and/or substitutions of one or more aminoacid residues. One type of alteration is an insertion, which involvesthe incorporation of one or more amino acids into a known peptide,polypeptide or protein structure. For ease of exposition, alterationswill be further described using a protein exemplar. Insertions may belocated at either or both termini of the protein, or may be positionedwithin internal regions of known proteins, which yield proteins such asfusion proteins and proteins having amino acid tags or labels.

Another type of alteration is a deletion, wherein one or more amino acidresidues in a protein are removed. Deletions can be effected at one orboth termini of the protein, or with removal of one or more residueswithin the amino acid sequence. Deletion alterations, therefore, includeall fragments of a protein described herein.

Yet another type of alteration is a substitution, which includesproteins wherein one or more amino acid residues are removed andreplaced with alternative residues. In one aspect, the substitutions areconservative in nature; however, the invention embraces substitutionsthat are also non-conservative. Conservative substitutions for thispurpose may be defined as set out in Tables A or B, below.

Amino acids can be classified according to physical properties andcontribution to secondary and tertiary protein structure. A conservativesubstitution is recognized in the art as a substitution of one aminoacid for another amino acid that has similar properties. Exemplaryconservative substitutions are set out in Table A as described inLehninger, [Biochemistry, 2nd Edition; Worth Publishers, Inc. New York(1975), pp. 71-77] and set out immediately below.

TABLE A Conservative Substitutions I SIDE CHAIN CHARACTERISTIC AMINOACID Non-polar (hydrophobic): A. Aliphatic A L I V P B. Aromatic F W C.Sulfur-containing M D. Borderline G Uncharged-polar: A. Hydroxyl S T YB. Amides N Q C. Sulfhydryl C D. Borderline G Positively charged (basic)K R H Negatively charged (acidic) D E

Alternative, exemplary conservative substitutions are set out in TableB, immediately below.

TABLE B Conservative Substitutions II ORIGINAL RESIDUE EXEMPLARYSUBSTITUTION Ala (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln,His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H)Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu (L) Ile, Val,Met, Ala, Phe Lys (K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe (F) Leu,Val, Ile, Ala Pro (P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y)Trp, Phe, Thr, Ser Val (V) Ile, Leu, Met, Phe, Ala

The binding site of HveA has been reported to be at the amino terminaldomain of gD (Carfi A., et al., 2001) The precise binding sites of gDfor Nectin 1 are not known, although it has previously been reported toinvolve gD amino acids 38 and 221 (Manoj S., et al., 2004; Zago A., etal., 2004; Connolly S A., 2005). Accordingly, in one aspect theinvention relates to amino acid alterations in the N-terminal region ofgD such that the ability of gD to bind HveA or Nectin 1 is reduced oreliminated A “natural receptor” as used herein is a cell surfacemolecule that interacts with wild-type HSV in the absence of humanintervention. For example, gB and gC of HSV-1 interact with heparansulfate proteoglycans in a natural infection. In preferred embodiments,gB and/or gC are altered to reduce or eliminate binding to heparansulfate proteoglycans. As another example, gD is known to bind toseveral receptors, including HveA and Nectin-1, in a natural infection.In preferred embodiments, gD is altered to reduce or eliminate bindingto HveA and/or Nectin-1.

Receptor-Ligands

As used herein, “receptor” and “ligand” refer to two members of aspecific binding pair and, hence, are binding partners. A receptor isthat member of the pair that is found localized on the surface of thecell; the ligand is the member of the pair that is found on the surfaceof HSV. Thus, in certain embodiments, the “ligand” may actually be whatthe art recognizes as a receptor outside the context of the inventionand the “receptor” may be its respective ligand. More generally, theinvention comprehends an HSV exhibiting a member of a binding pair, or afragment thereof that retains the capacity to specifically bind theother member of the binding pair, on its surface and the other member ofthat binding pair, or a fragment thereof that retains the capacity tospecifically bind its partner, is present on the surface of a targetcell.

One advantage of the invention is the ability to tailor HSV to target aspecific receptor while maintaining infectivity of the virus. In anexemplary embodiment, an HSV particle contains a fusion proteincomprising a portion of gD and the cytokine IL-13. Such a virus is ableto infect cells expressing the receptor IL-13Rα2. Because IL-13Rα2 isexpressed on the surface of cells of malignant gliomas, HSV containingthe gD/IL-13 fusion protein are effectively targeted to such cells.Ligands that bind to receptors which are overexpressed or differentiallyexpressed on either tumor cells or cells associated with tumor growth(e.g., neovasculature) are particularly preferred. Examples include theα_(v)β₃-α_(v)β₅ integrins, which are overexpressed in tumorneovasculature; epidermal growth factor receptor (EGFR), which isoverexpressed in head, neck, lung, colon, breast, and brain cancercells; HER-2/Neu, which is overexpressed in breast cancer cells; MUC-1,which is overexpressed in breast, lung, and pancreas cancer cells; andprostate-specific membrane antigen, which is overexpressed in prostatecancer cells. In certain embodiments, the ligand is a single-chainantibody that binds to its cognate specific binding pair member, hereinreferred to as a receptor. Single-chain antibodies have been shown to beeffective in targeting applications, as evidenced by their ability totarget retroviruses to specific receptors.

Essentially any two binding pair members or partners may be used asreceptor-ligands in the invention. However, it is contemplated thatcertain factors, such as the distance from the binding site on thereceptor to the membrane, or the conformation of the ligand when fusedto gD, may affect the efficiency of recombinant HSV fusion to the cellmembrane. Therefore, screens for effective receptor-ligand pairs arecontemplated, using no more than routine procedures known in the art.Additional screens, conventional in nature, may be used to optimizeconstructs. One routine method of screening is to follow the protocolprovided in the example for candidate receptor/ligand pairs, usingIL-13/IL-13 as a control receptor/ligand pair.

Alternatively, one may use a membrane fusion assay as described inTurner et al., 1998, incorporated herein by reference in its entirety.In the Turner assay, cells transfected with construct(s) encoding gB,gH, gL, and the gD/ligand fusion protein, and cells expressing thereceptor, are co-cultured and the cells are examined for membranefusion. Membrane fusion between gD/ligand-expressing cells andreceptor-expressing cells indicates that the candidate receptor-ligandpair (the ligand being a gD/ligand fusion protein) is functional.Constructs encoding functional gD/ligand proteins can then be used tocreate recombinant HSV that are targeted to cells expressing thereceptor.

Cell Targeting

Evident from the preceding discussion, another aspect of the inventionis the targeting of a recombinant HSV to a cell having a specificreceptor on its surface. In preferred embodiments, a recombinant HSV isdesigned to comprise a ligand that interacts with a receptor known to beexpressed on a cell of interest. The cell of interest is then infectedwith recombinant HSV. Such targeting methods may be used for a varietyof purposes.

In one aspect, a recombinant HSV is used to introduce a heterologousgene into a cell that expresses the receptor. In preferred embodiments,the cell is not infected by, or is poorly infected by, wild-type HSV.Thus, in certain embodiments, the invention provides a vector fortransforming a cell of interest with a heterologous gene.

Further, a cell can be rendered a target of a recombinant HSV of theinvention. The cell can be rendered a target by transforming the cell toexpress one member of a binding pair, e.g., a receptor capable ofspecifically binding a ligand expressed on a recombinant HSV. Forexample, as described in Example 2, the J1.1 cell line, which wasresistant to infection by a recombinant HSV expressing an IL-13 ligand,was rendered susceptible to infection by transforming the cell line witha vector encoding IL12Rα2 to produce the cell line J13R.

Generally, the targeted HSV according to the invention exhibit onemember of a binding pair, with the other member of that pair found onthe surface of a target cell. In some embodiments of the invention,targeting is achieved with a ligand-receptor binding pair, with theligand exhibited on the targeted HSV and the cognate receptor found onthe surface of the target cell, as described above. Although theinvention comprehends embodiments involving binding pairs that do notexhibit a ligand-receptor relationship (e.g., biotin-avidin) andembodiments in which the receptor is exhibited by the targeted HSV (the“receptor” defined above as a “ligand” using an alternative definitionof “ligand”) while the cognate ligand is found on the target cell (the“ligand” defined above as a “receptor” using an alternative definitionof “receptor”), embodiments in which the targeted HSV exhibits a ligandand the target cell presents the cognate receptor on its surface is usedas an illustrative embodiment to reveal the versatility of the inventionand to disclose the full scope thereof. For example, several ligandshave been used for receptor-mediated polynucleotide transfer. Someligands that have been characterized are asialoorosomucoid (ASOR) andtransferrin (Wagner et al., Proc. Natl. Acad Sci. USA, 87(9):3410-3414,1990). A synthetic neoglycoprotein, which recognizes the same receptoras ASOR, has also been used in a polynucleotide delivery vehicle (Ferkolet al., FASEB J., 7:1081-1091, 1993; Perales et al, Proc. Natl. Acad.Sci., USA 91:4086-4090, 1994) and epidermal growth factor (EGF) hasfurther been used to deliver polynucleotides to squamous carcinoma cells(Myers, EPO 0273085). Each of these specific approaches, and otherapproaches known in the art to achieve some selectivity in DNA delivery,or targeting, are amenable to use in the compositions and methods of theinvention and are contemplated as embodiments of the invention.

For embodiments in which a targeted HSV harboring a coding region, e.g.,a therapeutic coding region or gene, is delivered to a target cell, thenucleic acid encoding the therapeutic gene product may ultimately bepositioned and expressed at different sites. In certain embodiments, thenucleic acid encoding the therapeutic polynucleotide may be stablyintegrated into the genome of the cell. This integration may place thegene in its native location and orientation via homologous recombination(gene replacement) or it may be integrated in a random, non-specificlocation (gene augmentation). In yet further embodiments, the nucleicacid may be stably maintained in the cell as a separate, episomalsegment of DNA. Such nucleic acid segments or episomes encode functionssufficient to permit maintenance and replication independent of or insynchronization with the host cell cycle. How the expression constructis delivered to a cell and where in the cell the nucleic acid remains isdependent on the type of expression construct employed, as would beunderstood in the art.

It is envisioned that promoters subject to cell cycle regulation will beuseful in the present invention. For example, in a bicistronic HSVvector designed to treat a disease, disorder or condition by killing atarget cell, use of a strong CMV promoter to drive expression of a firstgene, such as p16, that arrests a cell in the G1 phase is accompanied byexpression of a second gene, such as p53, under the control of apromoter that is active in the G1 phase of the cell cycle, thusproviding a dual-gene approach to ensure that the target cell undergoesapoptosis. Other promoters, such as those of various cyclins, PCNA,galectin-3, E2F1, p53, BRCA1, and, indeed, any suitable promoter orexpression element known in the art, could be used.

In embodiments of the invention designed to treat diseases, disorders,or conditions associated with unwanted or excessive cell proliferation,such as cancer or restenosis, HSV is targeted to proliferating cellsthereby killing the cells. Because HSV is lethal to infected cells,expression of a heterologous gene is not required. However, inembodiments wherein the lethality of HSV is attenuated, an HSV harboringa gene that is lethal to the infected cell or that preventsproliferation of the infected cell may be used to target a cell.

Alternatively, HSV targeted to specific surface markers can be used tovisualize the distribution of tumor cells in tissues. This diagnostictool had been unavailable because of the indiscriminate binding of HSVto cells. Modification of HSV by eliminating (ablating) or reducing theindiscriminate binding of HSV to heparan sulfate proteoglycans withoutdeleteriously affecting the capacity of such HSV to replicate in bothdividing and non-dividing cells makes possible the use of these modifiedviral forms to visualize the distribution of tumor cells.

In one preferred method for visualizing the distribution of tumor cells,radioactive visualization is achieved by viral thymidine kinase(TK)-dependent incorporation of a radioactive precursor. Methods ofmolecular imaging of gene expression are well known in the art. Methodsoften use highly sensitive detection techniques such as positronemission tomography (PET) or single-photon emission-computed tomography(SPECT). In one embodiment, TK expression is measured using agancyclovir analog, such as9-(3-[¹⁸F]fluoro-1-hydroxy-2-propoxy)methylguanine, as the tracer ormarker (Vries et al., 2002). For a review of imaging TK gene expressionusing PET or SPECT, see Sharma et al., 2002 or Vries et al., 2002.

A second preferred imaging method is to fuse a non-critical tegumentprotein (e.g. U_(S)11, which is present in nearly 2000 copies per virusparticle) to a marker protein, such as green fluorescent protein, whichis capable of being visualized in vivo. Alternatively, a non-criticalprotein can be fused to a luciferase and the presence of the luciferasevisualized with a luminescent or chromatic luciferase substrate.Although a marker protein can be fused to essentially any viralstructural protein, preferred viral proteins include gC, gE, gI, gG, gJ,gK, gN, U_(L)11, U_(L)13, U_(L)14, U_(L)21, U_(L)41, U_(L)35, U_(L)45,U_(L)46, U_(L)47, U_(L)51, U_(L)55, U_(L)56, U_(S)10, and U_(S)11. Themarker protein also may be fused to thymidine kinase (Soling et al.,2002).

Library Screening

As noted above, HSV comprising a gD/ligand fusion protein can bind andinfect cells expressing a receptor to the ligand. In one embodiment, acell line expressing a receptor is used in screening for ligands of thereceptor. cDNA from a cDNA library is cloned into a vector encoding aportion of gD to produce a gD/cDNA-encoded fusion protein. The resultingvectors are then screened for membrane fusion using the assay of Turneret al. described above or by creating recombinant HSV expressing thegD/cDNA-encoded fusion protein and screening the viruses for the abilityto infect receptor-expressing cells. Such methods may be used, e.g., toidentify a ligand to an orphan receptor.

In other embodiments, mutations in, or variants of, the receptor orligand are screened to determine whether the mutants or variantsmaintain the ability to interact with the respective partner. Suchmethods may be useful in determining the specific residues important inreceptor-ligand interaction.

Therapeutic Methods

Another aspect of the invention is the use of the targeted HSV intherapeutic methods. By altering the cell-binding and infectivityproperties of the virus, many routes and methods of administrationbecome viable. For example, non-targeted HSV will bind indiscriminatelyto a variety of cells. Because of this property, large virus numbers areused and intravenous administration is generally not effective. However,by targeting the virus, one may lower the viral load (i.e., quantity ofvirus), yet maintain or increase efficacy. Furthermore, the targeted HSVcan be administered intravenously and produce therapeutic effects.

Therapeutic methods of the invention include those methods wherein anHSV is targeted to a receptor of a cell that contributes to, or is thebasis of, a disease or disorder. These targeted HSV can either exploitthe therapeutic properties of HSV itself (e.g., the lethality of HSV toinfected cells) or the targeted HSV can serve as a vector for thetargeted delivery of at least one therapeutic polynucleotide, such as anexpressible polynucleotide comprising a coding region. For example, inmethods wherein the targeted HSV contains one or more gene products thatrender the virus toxic to the cell or that prevent or inhibit cellproliferation, a preferred receptor is overexpressed or selectivelyexpressed on harmful or undesirable cells, such as cancer cells. Inother embodiments, the targeted HSV encodes a gene product that providesa desired function or activity in the targeted cell, e.g., when a cellhas one or more genetic defects preventing the cell from functioningproperly.

Additionally, it is contemplated that a therapeutic polynucleotide(e.g., gene or coding region) of a targeted HSV may be engineered to beunder the expression control of a cell- or tissue-specific expressioncontrol element, e.g., a promoter. In such embodiments, the targeted HSVprovide a further enhancement to the selective treatment of a suitabledisorder, disease or condition. The targeted HSV is specific for abinding partner located on the surface of those cells for whichtreatment is intended, and expression of the therapeutic coding regionor gene borne by the targeted HSV is limited to particular cells ortissues.

As HSV has been engineered to overcome the barriers to vector-basedtherapies, the choice of recombinant polynucleotide to be inserted intothe vector has widened to the point where a wide variety of diseases,disorders and conditions are amenable to treatment with targeted HSV. Anumber of diseases are amenable to polynucleotide-based therapy usingHSV (see, e.g., Kennedy, et al. Brain. 120, 1245-1259, 1997,incorporated by reference herein in its entirety). Though most attentionhas focused on cancers, there has been success in treating Parkinson'sdisease by expressing tyrosine hydroxylase in striatal cells, thusrestoring L-dopa-induced nerve repair following axotomy of the superiorcervical ganglion. Injection of a vector expressing nerve growth factorresulted in restored levels of tyrosine hydroxylase. More generally, HSVcan now be used in polynucleotide-based therapy to replace missing ordefective coding regions in the target cells. In the event of aninherited single-gene disorder (such as Lesch-Nyhan syndrome) where thecomplete DNA sequence, cause, and effect of the disorder are known, asingle polynucleotide replacement mediated by targeted HSV isappropriate and contemplated. Another strategy amenable to the use oftargeted HSV is the enhancement of endogenous expression levels of agene product, e.g., a growth factor or enzyme. Yet another strategy forusing targeted HSV is HSV-directed enzyme pro-drug therapy. The deliveryof a drug-sensitivity gene would be beneficial in the treatment of,e.g., a malignant brain tumor, making the tumor more susceptible toconventional anti-cancer agents.

In other embodiments, the targeted HSV of the invention provide forvector-mediated delivery of anti-sense oligodeoxyribonucleotides(oligonucleotides). The oligonucleotides, short segments of DNA (e.g.,2-100 nucleotides in length), are delivered to target cells and thereinbind to complementary mRNA, thus blocking the expression of specificgenes within the target cells. The encoded protein fail to besynthesized, as the MRNA is not be recognized by the translationalcomponents of the cell. In preferred embodiments, a deleterious gene istargeted.

In yet other embodiments, targeted HSV are used to deliverpolynucleotides, e.g., DNAs encoding gene products, that can recruit orenhance an immune system response, thereby bringing a subject's orpatient's own immune system to bear in the treatment of a disease,disorder or condition known in the art to be amenable to immune systemactivity. For example, an increase in cellular antigen expression oftumor cells, mediated by delivery of an expressible coding region forthe antigen by a targeted HSV, would enhance the immune response andincrease the susceptibility of such tumor cells to host cytotoxicimmunity.

In some embodiments, a targeted HSV composition of the invention isdelivered to a patient at or around the site of a tumor, which is a veryefficient method for counteracting clinical disease. Alternatively,systemic delivery of targeted HSV compositions may be appropriate inother circumstances, for example, where extensive metastasis hasoccurred, or where inaccessible tumors are encountered.

It is contemplated that in certain embodiments of the invention aprotein that acts as an angiogenesis inhibitor is targeted to a tumor.Also, an angiogenesis inhibitor agent may be administered in combinationwith a targeted HSV of the invention. These agents include, for example,Marimastat (British Biotech, Annapolis Md.; indicated for non-small celllung, small cell lung and breast cancers); AG3340 (Agouron, LaJolla,Calif.; for glioblastoma multiforme); COL-3 (Collagenex, Newtown Pa.;for brain tumors); Neovastat (Aeterna, Quebec, Canada; for kidney andnon-small cell lung cancer) BMS-275291 (Bristol-Myers Squibb,Wallingford Conn.; for metastatic non-small cell lung cancer);Thalidomide (Celgen; for melanoma, head and neck cancer, ovarian, andmetastatic prostate cancers; Kaposi's sarcoma; recurrent or metastaticcolorectal cancer (with adjuvants); gynecologic sarcomas, liver cancer;multiple myeloma; CLL, recurrent or progressive brain cancer, multiplemyeloma, and non-small cell lung, nonmetastatic prostate, refractorymultiple myeloma, and renal cancer); Squalamine (MagaininPharmaceuticals Plymouth Meeting, Pa.; non-small cell lung cancer andovarian cancer); Endostatin (EntreMEd, Rockville, Md.; for solidtumors); SU5416 (Sugen, San Francisco, Calif.; recurrent head and neck,advanced solid tumors, stage IIB or IV breast cancer; recurrent orprogressive brain (pediatric) cancer; ovarian cancer, AML (acute myeloidleukemia); glioma, advanced malignancies, advanced colorectal cancer,von-Hippel Lindau disease, advanced soft tissue cancer; prostate cancer,colorectal cancer, metastatic melanoma, multiple myeloma, malignantmesothelioma: metastatic renal, advanced or recurrent head and neckcancer, metastatic colorectal cancer); SU6668 (Sugen San Francisco,Calif.; advanced tumors); interferon-α; Anti-VEGF antibody (NationalCancer Institute, Bethesda Md.; Genentech San Franscisco, Calif., forrefractory solid tumors; metastatic renal cell cancer, in untreatedadvanced colorectal cancer; EMD121974 (Merck KCgaA, Darmstadt, Germany,for HIV-related Kaposi's sarcoma, and progressive or recurrentAnaplastic Glioma); Interleukin 12 (Genetics Institute, Cambridge,Mass., for Kaposi's sarcoma) and IM862 (Cytran, Kirkland, Wash., forovarian cancer, untreated metastatic cancers of colon and rectal origin,and Kaposi's sarcoma). The parenthetical information following theagents indicates the cancers against which the agents are being used inthese trials. It is contemplated that any of these disorders may betreated with the targeted HSV compositions of the invention, eitheralone or in combination with the agents listed.

In order to prepare a therapeutic composition for clinical use, it willbe necessary to prepare the therapeutic composition as a pharmaceuticalcomposition, i.e., in a form appropriate for in vivo applications.Generally, this will entail preparing compositions that are essentiallyfree of pyrogens, as well as other impurities that could be harmful tohumans or other vertebrates.

Generally, appropriate salts and buffers are included to render deliveryvectors stable and to allow for uptake by target cells. Aqueouscompositions of the invention comprise an effective amount of thetargeted HSV, dissolved or dispersed in a pharmaceutically acceptablecarrier or aqueous medium. Such compositions also are referred to asinocula. The phrase “pharmaceutically acceptable” or “pharmacologicallyacceptable” refer to molecular entities and compositions that do notproduce adverse, allergic, or other untoward reactions when administeredto an animal or a human. As used herein, “pharmaceutically acceptablecarriers” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Unless aconventional medium or agent is incompatible with either the vectors ofthe invention or the intended subject receiving treatment, its use intherapeutic compositions is contemplated. Supplementary active or inertingredients also can be incorporated into the compositions.

The active compositions of the invention include standard pharmaceuticalpreparations. Administration of these compositions according to theinvention is by any known route, provided that the target tissue isaccessible via that route. The pharmaceutical compositions may beintroduced into the subject by any conventional method, e.g., byintravenous, intradermal, intramusclar, intramammary, intraperitoneal,intrathecal, retrobulbar, intravesicular, intrapulmonary (e.g., termrelease); sublingual, nasal, anal, vaginal, or transdermal delivery, orby surgical implantation at a particular site. The treatment may consistof a single dose or a plurality of doses over a period of time.

Upon formulation, solutions are administered in a manner compatible withthe dosage formulation and in such amount as is therapeuticallyeffective. Appropriate dosages may be ascertained through the use ofestablished routine assays. As studies are conducted, furtherinformation will emerge regarding optimal dosage levels and duration oftreatment for specific diseases, disorders, and conditions.

In preferred embodiments, the unit dose may be calculated in terms ofthe dose of viral particles being administered. Viral doses are definedas a particular number of virus particles or plaque forming units (pfu).Particular unit doses include 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰,10¹¹, 10¹², 10¹³ or 10¹⁴ pfu. Particle doses may be somewhat higher (10-to 100-fold) due to the presence of infection-defective particles, whichis determinable by routine assays known in the art.

The pharmaceutical compositions and treatment methods of the inventionare useful in the fields of human medicine and veterinary medicine.Thus, the subject to be treated may be a vertebrate, e.g., a mammal,preferably human. For veterinary purposes, subjects include, forexample, farm animals such as cows, sheep, pigs, horses and goats,companion animals such as dogs and cats, exotic and/or zoo animals,laboratory animals including mice, rats, rabbits, guinea pigs andhamsters; and poultry such as chickens, turkey, ducks and geese.

In some embodiments of the invention, it is contemplated that thetargeted HSV is administered in conjunction with chemo- orradiotherapeutic intervention, immunotherapy, or with any other therapyconventionally employed in the treatment of cancer.

To kill cells, inhibit cell growth, inhibit metastasis, inhibitangiogenesis or otherwise reverse or reduce malignant phenotypes usingthe methods and compositions of the invention, one contacts a “target”cell, a tumor, or its vasculature with a targeted HSV composition and atleast one other agent. The components of these compositions are providedin a combined amount effective to kill or inhibit proliferation ofcancer cells. This process may involve contacting the cells with thetargeted HSV composition and the agent(s) or factor(s) at the same time.This may be achieved by contacting the subject organism, or cell ofinterest, with a single composition or pharmacological formulation thatincludes both agents, or by contacting the cell with two distinctcompositions or formulations, at the same or different times, whereinone composition includes a targeted HSV composition of the invention andthe other composition includes the second agent.

Another aspect of the invention provides diagnostic methods that involveimaging a tumor or diseased tissue using a targeted HSV. Such methodsare useful in diagnosing a patient with a disease, disorder, orcondition that is indicated by the presence of a receptor on the surfaceof a cell. Diagnostic imaging methods are discussed above.

Kits

Kits according to the invention may include recombinant viruses of theinvention or may include vectors for producing such recombinant viruses.A vector for producing a recombinant virus of the invention may encodethe gD/ligand fusion protein or may be designed to facilitate cloning ofa ligand to produce a gD/ligand fusion protein (e.g., a vectorcontaining a multiple cloning site within the gD coding region thatfacilitates in-frame insertions).

Other components that can be included in a kit of the invention includea receptor-expressing cell line (useful as a control), a nucleic acidmolecule for expressing the receptor in a particular cell type, andinstructions for using the kit to effect diagnostic analyses ortherapeutic treatments. In certain embodiments, a therapeutic kit willfurther contain a component for bringing about a therapeutic effect,such as a prodrug or a toxic compound. In other embodiments, adiagnostic kit will contain a compound useful in imaging methods, suchas a chromophore or fluorophore, or an antibody for detecting infectedcells.

Having provided a general description of the various aspects of theinvention, the following disclosure provides examples illustrative ofthe invention, wherein Example 1 describes construction of a targetedHSV, Example 2 illustrates the construction of a cell line expressing atargeted HSV, and Example 3 describes the controlled infection of adesired cell by a targeted HSV.

EXAMPLE 1 Construction of HSV targeting Vector R5111

A targeted HSV was constructed to target the recombinant virus to cellsof malignant gliomas. The target for entry of the virus into such cellsis the IL13Rα2 receptor known to be present in malignant gliomas. Unlikethe more prevalent IL13αR1 receptor, the IL13Rα2 receptor has a shortercytoplasmic domain and does not interact with IL4, of which IL13 is aclose relative. In general, the construction of the targeted HSVinvolved mutagenizing gB and gC to preclude their interaction withheparan sulfate. Also, IL13 was inserted into gD at amino acid 24thereby disrupting the gD binding site for HveA. The resulting IL13-gDchimeric virus can use IL13Rα2 for entry into cells carrying thatreceptor.

More specifically, the targeted HSV R5111 was constructed in severalsteps depicted in the four panels of FIG. 1 and detailed below.

(i) Substitution of Amino Terminal Domain of gC with IL13 Fused to theSignal Sequence of gC.

FIG. 1A, lines 1-3 schematically depicts a cDNA consisting of the IL13coding sequence fused at its amino terminus to its signal sequence. Thecomplete cDNA of IL 13, with the N-terminal signal peptide codingregion, was amplified using the PCR primer elongation method. Theprimers were as follows:

pIL13F1, (SEQ ID NO: 1) CATTGCTCTCACTTGCCTTGGCGGCTTTGCCTCCCCAGGCCCTGTGC-CTCCCTCTAGAGC; pIL13F2, (SEQ ID NO: 2)GCAGCTAGCCTCATGGCGCTTTTGTTGACCACG- GTCATTGCTCTCACTTGCCTTGGCGGC; andpIL13REcoRI, (SEQ ID NO: 3) GAGCTCGGATCCTGAATTCAACCGTCCCTC.

First-round PCR used pIL13F1 and pIL13REcoRI as primers, with pRB5830(containing the IL13 coding region) as the template. The PCR reactionmixture was then diluted 10-fold and 1 μl of the diluted reactionmixture was used as template for the second round of PCR amplificationswith pIL13F2 and pIL13REcoRI as the primer set. The PCR product wasgel-purified, digested with NheI/EcoRI, and ligated into pBluescript IIKS(+) at XbaI/EcoRI sites to generate pRB5832. To construct the transferplasmid pRB5835, a 4.8-kbp HindIII/SacI fragment containing the HSV-1 gCcoding region was released from cosmid pBC1007 and inserted intopBluescript II KS(+) to generate pRB5833. pRB5833 was cleaved with NheIand EcoRI and the N-terminal 148 residues of gC were replaced with thegC-signal/IL13 chimeric sequence (pRB5834). The insert in pRB5834 wasreleased by XhoI/SacI digestion and ligated into pKO5Y at the same sitesto generate pRB5835.

The recombinant virus R5107 (FIG. 1A, line 1) carrying the IL13-gCchimera was generated with the aid of the BAC-HSV system. RR1 competentcells that harbored bacterial artificial chromosome (BAC)-HSV bacmidswere transformed with the transfer plasmid pRB5835 by electroporation.After incubation for 1 hour at 30° C. in LB broth, the transformedbacteria were plated on pre-warned Zeocine (Zeo) plus chloramphenicol(Cm) (20 μg/ml of each) plates and incubated overnight at 43° C. forintegration. The next day, six colonies were picked and each wasseparately diluted in 1 ml LB. Five μl of the diluted bacteria were thenplated on Cm/10% sucrose (Suc) plates, and incubated at 30° C.overnight. To further confirm the loss of the replacement vector, 24Cm/Suc-resistant colonies (four colonies from each plate) wererestreaked in duplicate on Cm LB and Zeo LB plates, respectively. TheSuc^(r)/Cm^(r)/Zeo^(r) colonies were further screened by PCR (95° C., 4minutes for cycle 1; then 35 cycles of 94° C., 1 minute; 60° C., 1minute; and 72° C., 1 minute). The primers were:

pgC-F, GACACGGGCTACCCTCACTATCGAGGGC(SEQ ID NO: 4; from nt 96158 to 96185 in HSV-1 strain 17), and pgC-R,GGTGATGTTCGTCAGGACCTCCTCTAGGTC(SEQ ID NO: 5; from nt 96859 to 96830 in HSV-1 strain 17).

The DNA fragment amplified from PCR-positive clones (FIG. 2B) wassequenced to further confirm the integration of IL13 in the correct openreading frame (ORF) of gC. To verify the viability of the recombinant(R5607), the recombinant BAC-HSV DNA was prepared as described elsewhere(Ye et al., 2000) and transfected into rabbit skin cells byLipofectamine reagent (Life Technologies, Grand Island, N.Y.). Theresultant virus, R5607, was stored at −80° C.

(ii) Deletion of the Polylysine Track from gB, FIG. 1 Panel B.

To make a transfer plasmid for the deletion of the gB heparan sulfatebinding domain (polylysine), a 4.76 kbp BstEII fragment (from nt 53164to 57923 of HSV-1) containing the U_(L)27 (gB) ORF released from cosmidBC1014 was blunt-ended and cloned into pBluescript II KS (+) at a SmaIsite to generate pRB5846. To construct pRB5847, from which the10-amino-acid polylysine domain of gB was deleted, two fragmentsflanking the polylysine domain were amplified by PCR from pRB5846. Theprimer sets were:

pgB1BamHI: GTTCTTCTTCGGTTTCGGATCCCCCG; (SEQ ID NO: 6) pgB2BspEI:CGGCATTTCCGGAATAACGCCCACTC; (SEQ ID NO: 7) and pgB3BamHI:CAGAAAACCGGATCCCCCAAAGCCGCC; (SEQ ID NO: 8) pgB4BsiWI:GCCAACACAAACTCGTCGTACGGGTAC. (SEQ ID NO: 9)

PCR amplified fragments were then cut with BspEI/BamHI, or BsiWI/BamHIand ligated into pRB5846, which had the 1.2 kbp BsiWI/BspEI fragmentalready deleted. To generate the transfer plasmid pRB5848, the 4.76 kbpinsert in pRB5847 was released by XbaI/EcoRV digestion and ligated intopKO5Y at the sites of XbaI and ScaI. Recombinant HSV-1 virus R5108 isbased on R5107 with the additional deletion of the gB heparan sulfatebinding domain. It was made by the same procedure as BAC-R5607, exceptthat the transfer plasmid pRB5848 was used instead of BAC-HSV wild-typeand pRB5835. The sequence of the mutant gB was verified by sequencingthe entire ORF.

(iii) Deletion of gD (FIG. 1 Panel C, Lines 6 and 7).

The coding sequence of gD was replaced with the human cytomagolovirusimmediate early promoter to enable the expression of glycoprotein I. A0.65 kbp fragment containing the promoter was released from pRB5836 byClaI digestion and inserted into pgD⁻ (FIG. 6), a plasmid obtained fromG. Campadelli-Fiume. This plasmid, containing the flanking sequences ofgD but lacking the gD ORF, had been cut with ClaI to generate pRB5849.pRB5849 was then cut with NotI and PmeI and ligated into pKO5Y at theNotI and ScaI sites to generate the transfer plasmid pRB5850.Recombinant HSV-1 virus R5110 is based on R5608 with the additionaldeletion of gD. It was made by the same procedure as BAC-R5607 exceptthat transfer plasmid pRB5850 was used instead of BAC-HSV wild-type andpRB5835. The recombinant BAC-HSV DNA was prepared as described in (Ye etal., 2000). The mutant virus was designated R5110.

(iv) Construction of the R5111 Mutant Carrying the IL-13-gD ChimericGene (FIG. 1 Panel D).

Plasmid pRB123 carries a 6,584 bp BamHI J fragment containing the gDcoding region and flanking sequences in the BamHI site of pBR322. Toconstruct the IL 13-gD chimeric plasmid, pRB123 was digested with AflIIand HpaI to release two fragments of 7.6 kb and 3.2 kb. The 3.2 kbfragment was further digested with FspI to release 2.5 kb and 0.7 kbfragments that contain the amino-terminal 661 bp of the gD ORF. Apolylinker sequence containing the restriction sitesXhoI-BglII-EcoRI-KpnI was inserted into the 0.7 kb fragment downstreamof the 24th codon of gD by two PCR reactions using a first forwardprimer,

(SEQ ID NO: 10) 5′-CAGTTATCCTTAAGGTCTCTTTTGTGTGGTG-3′,and a first reverse primer,

(SEQ ID NO: 11) 5′-CCGGAATTCCGGAGATCTTCCCTCGAGGACCGGAAGGTCTTTGCCGCGAAAG-3′,and a second forward primer,

(SEQ ID NO: 12) 5′CCGGAATTCCGGGGTACCCTGGACCAGCTGACCGACCCTCCGG-3′,and a second reverse primer,

(SEQ ID NO: 13) 5′-CGGGGGGATGCGCAGCGGGAGGGCGTACTTAC-3′,

respectively. After digestion of the two PCR products by EcoRI, theywere ligated and amplified by PCR again to obtain the desired DNAfragment containing the polylinker insertion.

IL13 was amplified by PCR with the forward primer,

(SEQ ID NO: 14) 5′-CCGCTCGAGATGGCGCTTTTGTTGACCACGG-3′,and the reverse primer,

5′-GGGGTACCGTTGAACCGTCCCTCGCGAAA-3′, (SEQ ID NO: 15)

and then inserted into the XhoI and KpnI sites of the 0.7 kb fragmentdescribed above. This new fragment with the IL13 insertion was thenligated with the 2.5 kb and 7.6 kb fragments (see above) to generate theIL13-gD chimeric transfer plasmid, pRB13-24.

R5111 was generated by co-transfection of transfer plasmid pRB13-24 andthe R5110 viral DNA into U87 glioma cells. The progeny of thetransfection was plated at a high dilution on Vero and HEp-2 cellcultures to yield individual, well-spaced plaques. From each of theinfected cell cultures, six single plaques were picked, frozen-thawed,sonicated, and then replated on fresh cultures of Vero or HEp-2 cells(depending on the origin of the plaque) for preparation of virus stocksand to prepare viral DNA for sequencing.

Viral DNA Extraction.

Infected cells were removed from each of the 25 cm² flasks exposed toindividual plaque isolates, rinsed, and resuspended in 500 μl ofLyse-O-Lot (150 mM NaCl, 10 mM Tris, 1.5 mM MgCl₂ in the presence of0.1% of NP40). Nuclei were removed by low-speed centrifugation. To thesupernatant fluid were added sodium dodecyl sulfate (SDS) to 0.2%, EDTAto 5 mM and β-ME to 50 mM. The solution was then extracted twice withphenol/chloroform. Viral DNA was finally precipitated by ethanol,resuspended, and the IL13 ORF and IL13-gD chimeric reading frame wereamplified by PCR with two sets of primers. The first set, designed toamplify IL13, consisted of: a forward primer,5′-CCGCTCGAGATGGCGCTTTTGTTGACCACGG-3′ (SEQ ID NO:16), and a reverseprimer, 5′-GGGGTACCGTTGAACCGTCCCTCGCGAAA-3′ (SEQ ID NO:17), which willamplify the IL13 ORF. The second set, designed to amplify the IL13-gDjunction, consisted of a forward junction primer,5′-CCGCTCGAGATGGCGCTTTTGTTGACCACGG-3′ (SEQ ID NO: 18), and a reversejunction primer, 5′-AACTGCAGGTTGTTCGGGGTGGCCGGGGG-3′ (SEQ ID NO:19). All12 IL13-gD PCR products were sequenced to determine whether the gDsequence contained deletions or substitutions.

Verification of the Structure of R5111

The construction of the R5111 virus is depicted in FIG. 1. The designinvolved replacement of the HveA binding site with the IL13 ligand toenable the recombinant virus to bind the IL13α2 receptor on cellsurfaces and to delete the sequences reported to bind to heparansulfate. Verification of the structure of R5111 was done as follows:

(i) The replacement of the amino-terminal domain of gC with IL13 and thedisruption of the heparan sulfate binding site was initially verified bysequencing gC from recombinant R5107 (FIG. 2 A).

(ii) The deletion of codons 68-77 of gB was verified by sequencing thegB ORF amplified by PCR from recombinant R5108 (FIG. 2 B). Thenucleotide and amino acid sequences of gB with the polylysine trackdeleted are set out in SEQ ID NOs.:35 and 36, respectively.

(iii) The presence of chimeric IL13-gD in R5111 was verified by PCR, asillustrated in FIG. 3, and by sequencing the entire IL13-gD codingregion, amplified by PCR, as shown in FIG. 2. The nucleotide and aminoacid sequences of gD with the IL13 integration are set out in SEQ IDNOs.:39 and 40, respectively. The R5111 recombinant was initiallyisolated from transfected U187 cells and grown in parallel in Vero cellsand HEp-2 cells. To determine whether the virus grown in Vero cells orHEp-2 cells differed with respect to amino acid sequence, six plaqueseach from Vero or HEp-2 cultures containing well-separated plaques werepicked. In this series of verification experiments, two sets of primerswere used to confirm the presence of the IL13 insert in gD and to verifythe presence of a junction between IL13 and gD. In a second round ofverifications, the 12 clones of gD were sequenced to determine whetherthe isolates obtained from the viruses passaged in Vero cells or inHEp-2 cells differed in amino acid sequence. No differences were found.Furthermore, except for the inserted IL13 sequence, no differences werefound between the sequence of HSV-1 (F) gD and those of the clonedIL-13-gD chimeric genes (FIG. 2 C).

(iv) In denaturing polyacrylamide gels, IL13 migrated as a protein withan apparent Mr of 15-17,000. In the recombinant R5111, IL13 replaced 148amino acids of gC. FIG. 4B shows an immunoblot of electrophoreticallyseparated proteins from a lysate of R5111 mutant-infected cells exposedto an antibody to gC. As illustrated in that figure, the anti-gCantibody reacted with proteins present in lysates of HSV-1(F) and withproteins from R5111 lysates, exhibiting similar electrophoreticmobilities. In contrast, an antibody to IL13 reacted with a band ofsimilar mobility in R5111 lysates (FIG. 4C, lane 3) but not in lysatesof HSV-1(F) (FIG. 4 C, lane 2). The IL13-gD fusion protein in the R5111mutant virus was verified by reacting the cell lysates with gD and IL13antibody. Comparison of wild-type gD and the chimeric IL13-gD chimericprotein (FIG. 4 A, lane 3), showed that, as expected, IL13-gD migratedmore slowly than the wild-type gD (FIG. 4 A, lane 2). The fastermigrating band of gD did not react with the antibody to IL13 (FIG. 4C,lane 2).

EXAMPLE 2 Construction of a Cell Line Expressing the IL13 Receptor(IL13Rα2)

A rigorous test of the ability of R5111 to utilize the IL13Rα2 proteinas a receptor for entry required construction of a cell line expressingIL13Rα2 (nucleotides 126-1265 of SEQ ID NO.:33; SEQ ID NO:34) in theabsence of other HSV-1 entry receptors. The J1.1 cell line was selectedfor this construction. In essence, this cell line lacks the receptorsnecessary for the entry of virus into cells and the cell line is notsusceptible to infection by wild-type virus. The construction of aplasmid encoding a IL13Rα2 protein fused at its carboxyl terminus to aHA tag, transfection of J1.1 cells with the plasmid encoding the taggedIL13Rα2 protein, and selection of the cell line expressing the proteinis described below.

To test for the production of IL13Rα2 protein, five clones of theselected cells were harvested, solubilized, subjected to electrophoresisin denaturing polyacrylamide gels and tested for expression of theprotein.

Construction of J13R, a Cell Line Stably Expressing IL13Rα2 Receptor.

The IL13α2 coding region was tagged with an HA tag at its 3′ end (thecarboxyl terminus of the encoded polypeptide) by PCR with forwardprimer, 5′-AAGATTTGGGC-TAGCATGGCTTTCGTTTGC-3′ (SEQ ID NO:20), andreverse primer,5′-TCCCTCGAAGCTTCAAGCATAATCTGGCACATCATATGTATCACAGAA-AAA-3′ (SEQ IDNO:21). NheI and HindIII restriction digests were used to createcompatible ends. The DNA fragment was then inserted into pcDNA 3.1 (zeo)vector (Invitrogen; Carlsbad, Calif.) to generate transfer plasmidpRB13-R2. All of the constructs were sequenced to insure fidelity.

J1.1, a derivative of BHK thymidine kinase-cells which lack both HveAand nectin 1 receptors, was obtained from Dr. G. Campadelli-Fiume,University of Bologna, Italy. J10.1 cells, stably transfected with pRB13-R2 using a Lipofectamine kit (Gibco-BRL), were selected on the basisof their resistance to zeocin (Invitrogen). Zeocin-resistant clones wereamplified and screened for IL13Rα2 expression by immunoblotting withanti-HA polyclonal antibody. Lysates of parental and transformed cellsformed by solubilized in SDS were each electrophoretically separated ina denaturing gel (50 μg/lane), transferred to a nitrocellulose sheet,and probed with antibody against HA (Santa Cruz Biotechnology). Theprotein bands were visualized by an enhanced chemiluminescent detection(ECL) system (Pierce, Rockford, Ill.) according to the instructions ofthe manufacturer. One (J13R-2) was selected for testing the ability ofR5111 to use the IL13Rα2 receptor.

As shown in FIG. 5, all clones expressed a protein band reactive withthe anti-HA antibody. The apparent size of the protein was consistentwith the reported size of IL13Rα2. Of 5 J13R-positive clones, J13R-2(FIG. 5, lane 3) was selected and designated J13R.

EXAMPLE 3 Infection by the HSV Targeting Vector R5111

SK-N-SH, HEp-2, Vero, and U87 cells were obtained from American TypeCulture Collection (Rockville, Md.) and maintained in Dulbecco'smodification of Eagle's Minimal Essential Medium (DMEM) supplementedwith 10% fetal bovine serum. Replicate cultures of SK-N-SH, HEp-2, Vero,U87, J1.1, and J13R were exposed to 0.01 PFU of R5111 virus per cell.After 24 hours of incubation, the cells were harvested and viral yieldswere titered on Vero cells.

Immunoblotting Electrophoretically Separated Proteins.

The indicated cells were mock-infected or exposed to 10 PFU ofrecombinant or wild-type HSV-1(F) per cell. The cells were harvested at24 hours after infection, disrupted in SDS disruption buffer, boiled,cleared by centrifugation and electrophoretically separated on a 10%denaturing polyacrylamide gel. After transfer to a nitrocellulosemembrane, the isolated proteins were reacted with antibodies asindicated using known and conventional techniques. Monoclonal antibodiesagainst gD-(clone H170), gC- and HA-specific polyclonal antisera werepurchased from the Goodwin Institute, Plantation, Fla. Polyclonalantibodies against IL13 were purchased from Santa Cruz Biotechnology.

The results shown in Table 1 were as follows: R5111 replicated to withina 10-fold range in HEp-2, Vero, U87, and J13R cells. The titer obtainedfrom J1.1 cells was approximately 10⁵-fold lower than that obtained fromall other cell lines. To test whether the J13R cell line had acquired areceptor for wild-type HSV-1 (HSV-1(F)), J1.1 and J13R cells were alsoexposed to the wild-type virus. The results, also shown in Table 1,indicate that the cells remain resistant to the wild-type virus. It wasknown that HEp-2 cells express the nectin receptor but not the HveAreceptor. The results show that the targeted HSV containing an IL13-gDfusion can target (i.e., bind and infect) cells expressing a particularreceptor (IL13Rα2) approximately as well as wild-type HSV targets cellsexpressing the HveA receptor. The results indicate that R5111 can useIL13Rα2 as a receptor for entry in a cell line lacking all other HSV-1receptors.

TABLE 1 Replication of R5111 in various cell lines Virus Cell Line*Yield** R5111 Vero 11 × 10⁷ HEp-2 1.2 × 10⁷  SK-N-SH 17 × 10⁷ U87 27 ×10⁷ J1.1  2 × 10² J13R 11 × 10⁷ HSV-1(F) J1.1  6 × 10³ J13R  4 × 10³*cell lines derived from human brain tumors. **The cells were exposed to0.01 PFU of R5111 or HSV-1(F) per cell and harvested 24 hours afterinfection. Progeny virus were titered on Vero cells.

This disclosure contains an exemplary description of the constructionand properties of a recombinant HSV virus, R5111. In R5111, the heparansulfate binding sites on the surface of the viral particle were ablatedto preclude or at least reduce the attachment of virus to non-targetedcells. Attachment even in the absence of fusogenic activity may lead toendocytosis, degradation of the virus particle, and to potential damageto the cell by lysosomal enzymes (Zhou et al. 2002; Zhou et al. 2000).At the same time, a copy of IL13 was inserted into gC to enhance bindingof virus particles to the IL13Rα2 receptor. The major restructuring ofthe viral genome consisted of insertion of IL13 at amino acid 24 of gD.Available data indicate that this modification ablates the gD bindingsite for the HveA receptor (Carfi et al. 2001). The data obtained usingR5111 indicate that the virus retains the capacity to interact with theNectin receptor. Nonetheless, the R5111-targeted HSV was able to infectand replicate in J13R cells but not in the parental, J1.1, cells.

EXAMPLE 4 Construction of HSV Targeting Vector R5141 and R5144

A therapeutic herpes simplex virus 1 (HSV-1) capable of infecting andreplicating solely in cells harboring the IL13Rα2 receptor wasconstructed using recombinant DNA techniques. As disclosed above,construction of R5111, which expresses IL13 on its surface and lacks thebinding sites for heparin sulfate, allowed R5111 to infect J-13R cellsas well as cells exhibiting the natural receptors for HSV-1. Thus, theinvolvement of fusogenic glycoproteins of R5111—a key step in viralentry—is independent of the receptor with which gD interacts.

The binding site of HveA has been reported to be at the amino terminaldomain of gD (Carfi A., et al., 2001). The precise binding sites of gDfor Nectin 1 are not known, although it has previously been reported toinvolve gD amino acids 38 and 221 (Manoj S., et al., 2004; Zago A., etal., 2004; Connolly S A., 2005). The general assumption within the fieldis that the HveA and Nectin 1 sites do not overlap and that eachindependently promotes the same structural alteration of gD to enableentry of the virus into cells. However, it is possible that the surfacestructure of HveA and Nectin 1 at their binding sites may be similareven though the Hve1 and Nectin1 amino acid sequences are not identical.

Based in part on the foregoing information, viruses capable ofproductive replication solely in targeted cells were designed as shownin FIG. 7. Using standard molecular biological cloning techniques knownin the art, recombinant virus R5141 was constructed by inserting IL13 inthe place of gD residues 1-32. In addition, the valine residue atposition 34 was substituted with serine (“V34S”) (SEQ ID NOs.:41 and 42,respectively). Similarly, recombinant virus R5144 was constructed byinserting IL13 in the place of gD residues 1-32, and the valine atposition 37 was substituted with serine (“V37S) (SEQ ID NOs.:43 and 44,respectively).

One of skill would appreciate that a variety of re-targeted SHV, bothHSV-1 and HSV-2, could be constructed and assessed using routinetechniques in view of the disclosures herein. In particular,substituting a binding domain of a binding partner for the N-terminalregion of gD, e.g., for amino acids 1-32 of gD, would be within theskill in the art. Further alteration of the gD fusion, e.g., by aminoacid substitution, whether conservative substitution or not, would alsobe within the skill in the art. Of course, additions or deletions to gDfusions would also be within the skill in the art and assessing thetargeting capacities of such constructs would involve routineexperimentation in view of the teachings herein.

EXAMPLE 5 Infection by the HSV Targeting Vectors R5141 and R5144

The capacities of the recombinant viruses described in Example 4 toproductively replicate solely in targeted cells were assessed using thecell lines which express either HveA (J-HveA) alone (relative to thegroup of HveA, Nectin1, and IL13Rα2), Nectin1 (J-Nectin1) alone, orIL13Rα2 (J-13R) alone. For cell infection, the procedure set forth inExample 3 was repeated using recombinant virus R5141 and recombinantvirus R5144.

The replication of R5141 and R5144 in J-Nectin-, J-HveA-, andJ-13R-specific cells are summarized in Table 2. R5141 and R5144 do notproductively interact with either native gD receptors, HveA or Nectin1.Significantly, however, R5141 interacts with and replicates in IL13Rα2for productive entry into cells.

TABLE 2 Replication of R5141 and R5144 in J-Nectin, J-HveA and J- 13Rcells. HSV-1 (F) R5141 R5144 J-Nectin 4 × 10⁸ 7 × 10¹ 5 × 10¹ J-HveA 3 ×10⁸ 4 × 10¹ 3 × 10² J-13R 3 × 10¹ 5 × 10⁶ 7 × 10²

Thus, recombinant virus R5141 is capable of productive replicationsolely in targeted cells and this result opens the way for developmentof therapeutic viruses targeting cells exhibiting the IL13Rα2 receptor,such as malignant gliomas and other human tumors exhibiting IL13Rα2. Itis expected that other mutations (i.e., those that abolish binding ofNectin and those that have a similar effect on HveA) will yield virusesthat enter solely via non-natural HSV receptors.

EXAMPLE 6 HSV Targeting Vector R5161

An HSV targeting vector designated HSV R5161 has a structure analogousto the structure of HSV R5141, and HSV R5161 was constructed in themanner described in Example 4, above, with the exception that HSV R5161contains the sequence encoding the HSV gD leader sequence, whereas HSVR5141 contains the sequence encoding the IL-13 leader sequence. Inparticular, HSV R5161 encodes an IL-13-gD fusion protein in which IL-13sequence replaces the sequence encoding gD amino acids 1-32, with a V34Ssubstitution in the gD moiety of the fusion protein, as described abovein the context of describing HSV R5141.

The relative capacities of recombinant viruses HSV R5141 and HSV R5161to productively replicate in targeted cells were measured in the J-13Rcell line, which expresses IL13Rα2, but not HveA or Nectin1. HSV R5161was expressed at a level 10-fold higher than the expression level of HSVR5141 in J-13R cells.

It is expected that in the majority of embodiments of the invention,relatively high levels of expression will be advantageous in ensuringthat targeted cells are efficiently contacted by the re-targeted HSV. Inre-targeting the HSV, the invention provides an approach to controllingthe virulence of the virus in a manner that minimizes undesirablepathogenicity, i.e., pathogenicity towards non-targeted cells. Inaddition, the virulence of any re-targeted HSV can be further attenuatedusing known approaches to virulence control that do not interfere withthe re-targeting, such as by mutating the γ₁34.5 gene(s). In thoseembodiments in which it is desirable to have relatively high expressionlevels of the re-targeted HSV, it is preferred that the leader sequenceof gD be used. Alternative leader sequences, such as leaders from otherHSV genes, are contemplated. Moreover, expression control elements(e.g., promoters, enhancers, expression factor binding sites) can beengineered to achieve desired expression levels of the fusion proteinusing ordinary levels of skill and techniques known in the art.

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The invention claimed is:
 1. A recombinant herpes simplex virus (HSV)particle having at least one protein on its surface, comprising: (a) analtered gD, wherein the alteration reduces binding of gD to one or moreof its cellular receptors, said alteration comprising (i) a heterologouspeptide ligand on the surface of the recombinant HSV particle forming afusion protein with the altered gD, wherein a polynucleotide encodingthe fusion protein is joined to a heterologous coding region for aleader sequence; and (ii) an amino acid alteration; wherein saidrecombinant HSV particle preferentially binds to cells expressing abinding partner to said heterologous peptide ligand.
 2. The recombinantherpes simplex virus (HSV) particle of claim 1 further comprising analtered viral surface protein, wherein the alteration reduces binding ofthe viral surface protein to a sulfated proteoglycan.
 3. The recombinantHSV particle of claim 1, wherein the alteration is a conservative aminoacid substitution.
 4. The recombinant HSV particle of claim 1, whereinthe amino acid alteration is selected from the group consisting of anamino acid deletion, an amino acid substitution and an amino acidinsertion.
 5. The recombinant HSV particle of claim 4 wherein the aminoacid alteration is an amino acid substitution.
 6. The recombinant HSVparticle of claim 5 wherein the amino acid substitution is at position34.
 7. The recombinant HSV particle of claim 6, wherein the amino acidsubstitution is V34S.
 8. The recombinant HSV particle of claim 7,wherein the fusion protein encodes an IL-13-gD fusion and wherein theleader sequence is an HSV gD leader sequence.
 9. The recombinant HSVparticle of claim 1, wherein the leader sequence is an HSV leadersequence.
 10. The recombinant HSV particle of claim 9, wherein theleader sequence is an HSV gD leader sequence.
 11. The recombinant HSVparticle of claim 1, wherein the viral surface protein is selected fromthe group consisting of gB and gC.
 12. The recombinant HSV particle ofclaim 1, wherein the alteration of gD reduces binding to at least oneprotein selected from the group consisting of HveA and Nectin-1.
 13. Therecombinant HSV particle of claim 1, wherein the ligand forms a secondfusion protein with a viral surface protein selected from the groupconsisting of gB and gC.
 14. The recombinant HSV particle of claim 1,wherein the binding partner is a cell surface receptor for said ligand.15. The recombinant HSV particle of claim 14, wherein the cell is acancer cell.
 16. The recombinant HSV particle of claim 15, wherein thecancer cell is a malignant gliomal cell.
 17. The recombinant HSVparticle of claim 1, wherein the ligand is a cytokine.
 18. Therecombinant HSV particle of claim 17, wherein the cytokine is IL13. 19.The recombinant HSV particle of claim 1, wherein the ligand is asingle-chain antibody.
 20. A pharmaceutical composition comprising therecombinant HSV particle of claim 1 and a pharmaceutically acceptablecarrier, diluent, or excipient.
 21. A kit comprising the pharmaceuticalcomposition according to claim 20 and a set of instructions foradministering the composition to a subject in need.
 22. A method oftargeting a recombinant HSV particle to a cell comprising (a)identifying a ligand for a ligand binding partner exhibited on thesurface of a target cell; and (b) creating an HSV particle according toclaim 1, wherein the ligand binds to the binding partner exhibited onthe surface of said target cell.
 23. The method of claim 22, wherein asecond viral surface protein selected from the group consisting of gBand gC is altered to reduce binding of said second viral surface proteinto a sulfated proteoglycan.
 24. The method of claim 22, wherein thealteration to gD reduces binding of gD to at least one cellular receptorfor gD selected from the group consisting of HveA and Nectin-1.
 25. Themethod of claim 22, wherein the ligand forms a second fusion proteinwith gC.
 26. The method of claim 22, wherein the cell is a cancer cell.27. The method of claim 26, wherein the cancer cell is a malignantgliomal cell.
 28. The method of claim 25, wherein the ligand is acytokine.
 29. The method of claim 28, wherein the cytokine is IL13. 30.The method of claim 22, wherein the ligand is a single-chain antibody.31. A method of imaging a cell comprising: (a) contacting the cell witha recombinant HSV particle according to claim 1, said recombinant HSVparticle further comprising a coding region for a marker protein; and(b) detecting the presence of the marker protein.
 32. The method ofclaim 31, wherein the cell is a cancer cell.
 33. The method of claim 31,wherein the binding partner is present at a higher number on the cancercell as compared to a non-cancerous cell of the same type.
 34. Themethod of claim 31, wherein the marker protein is selected from thegroup consisting of thymidine kinase, green fluorescent protein, andluciferase.
 35. The method of claim 31, wherein the amino acidsubstitution is V34S.
 36. A method of treating a cell-based diseasecomprising delivering a therapeutically effective amount of arecombinant HSV particle according to claim 1 to a subject in need. 37.The method according to claim 36 wherein the disease is cancer.
 38. Amethod of ameliorating a symptom associated with a disease comprisingadministering a therapeutically effective amount of a recombinant HSVparticle according to claim 1 to a subject in need.
 39. The methodaccording to claim 38 wherein the disease is characterized byhyperproliferative cells.
 40. A method of delivering a therapeuticallyuseful peptide to a cell comprising: (a) inserting a coding region for atherapeutically useful peptide into the DNA of a recombinant HSVparticle according to claim 1, thereby producing a recombinant HSVclone; and (b) delivering said recombinant HSV clone to said cell.
 41. Amethod of killing a target cell, comprising contacting the target cellwith a recombinant HSV particle according to claim
 1. 42. The methodaccording to claim 22 wherein the amino acid alteration is an amino acidsubstitution.
 43. The method according to claim 42 wherein the aminoacid substitution is at position
 34. 44. The method of claim 43, whereinthe amino acid substitution is V34S.
 45. The recombinant HSV particle ofclaim 10, wherein the HSV gD leader sequence comprises amino acids 1-32of gD.
 46. The recombinant HSV particle of claim 1, wherein the fusionprotein is obtained by inserting IL-13 in the place of amino acids 1-32of gD.